Method for differentiation induction in cultured stem cells

ABSTRACT

Provided is a method of inducing the differentiation of a stem cell into nerve progenitor cells, comprising the step (1) of forming homogenous aggregates of stem cells in a serum-free medium (1) and the step (2) of suspension-culturing the homogenous aggregates of stem cells in the presence of a basement membrane reference standard.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of copending U.S. patentapplication Ser. No. 13/508,303, filed on Oct. 22, 2012, which is theU.S. national phase of International Patent ApplicationPCT/JP2010/070163, filed Nov. 5, 2010, which claims the benefit of U.S.Provisional Patent Application No. 61/258,439, filed Nov. 5, 2009, whichare incorporated by reference in their entireties herein.

TECHNICAL FIELD

The present invention relates to a method of differentiation inductionof stem cells, more specifically to a method of differentiationinduction of stem cells comprising combining quick re-aggregation andthree-dimensional suspension culture in performing stem cell aggregateculture.

BACKGROUND ART

To date, some culturing methods for differentiation induction of nervesfrom pluripotent stem cells such as ES cells have been known, includingthose reported by the present inventors [Watanabe, K., Ueno, M., Kamiya,D., Nishiyama, A., Matsumura, M., Wataya, T., Takahashi, J. B.,Nishikawa, S., Nishikawa, S.-i., Muguruma, K. and Sasai, Y. (2007), AROCK inhibitor permits survival of dissociated human embryonic stemcells. Nature Biotechnology 25, 681-686, Su, H.-L.; Muguruma, K.,Kengaku, M., Matsuo-Takasaki, M., Watanabe, K., and Sasai, Y. (2006),Generation of Cerebellar Neuron Precursors from Embryonic Stem Cells.Developmental Biology 290, 287-296; Ikeda, H., Watanabe, K., Mizuseki,K., Haraguchi, T., Miyoshi, H., Kamiya, D., Honda, Y., Sasai, N.,Yoshimura, N., Takahashi, M. and Sasai, Y. (2005), Generation ofRx+/Pax6+ neural retinal precursors from embryonic stem cells. Proc.Natl. Acad. Sci. USA 102, 11331-11336; pamphlet for WO2005/123902; andJP-A-2008-99662]. There are high expectations for ES cell-derived nervecells (e.g., dopamine nerve cells and the like) as a source of graftcells for cell transplantation therapy in regenerative medicine forintractable neurologic diseases. To this end, disease-related nervecells that are present in the brain and surrounding tissues must beproduced accurately. However, because an extremely large number of kindsof nerve cells are present in the brain and surrounding tissues, thereare still many types of nerve cells and tissues for which efficient invitro differentiation has been unsuccessful.

The retina, a component of the eyeball, derived from the diencephalon,occurs as a thin membranous tissue covering the inner wall behind theeyeball. Observed in the retina is a laminar structure of regularlyarranged nerve cells. Nerve cells of the retina can be roughly dividedinto five types: photoreceptor cells (cones, rods), bipolar cells,horizontal cells, amacrine cells, and ganglion cells. Light is convertedto an electric signal in photoreceptor cells; the signal (information)is transmitted to bipolar cells and horizontal cells via chemicalsynapses. Bipolar cells connect with amacrine cells and nerve ganglioncells via synapses, and the axons of ganglion cells, as optic nerves,communicate with the visual center of the cerebrum. For the treatment ofretinopathies, etiologic research, drug discovery research, celltransplantation therapy research and the like have been conducted sofar, but it is extremely difficult to obtain human retinal tissue forthe sake of such research. Although it has recently become possible toinduce the differentiation from induced pluripotent stem cells toretinal pigment epithelium [see Hirami Y, Osakada F, Takahashi K, OkitaK, Yamanaka S, Ikeda H, Yoshimura N, Takahashi M. (2009), Generation ofretinal cells from mouse and human induced pluripotent stem cells.Neurosci Lett. 458(3):126-31], it has been difficult to control theselective differentiation induction and genesis into particular retinalneurons and a retinal tissue containing the same.

The present inventors showed that dispersion suspension culture using aserum-free medium (the SFEB method) is effective as a method fordifferentiation induction of nerves from pluripotent stem cells such asanimal and human ES cells [see Ikeda, H., Watanabe, K., Mizuseki, K.,Haraguchi, T., Miyoshi, H., Kamiya, D., Honda, Y., Sasai, N., Yoshimura,N., Takahashi, M. and Sasai, Y. (2005), Generation of Rx+/Pax6+ neuralretinal precursors from embryonic stem cells. Proc. Natl. Acad. Sci. USA102, 11331-11336; Watanabe, K., Kamiya, D., Nishiyama, A., Katayama, T.,Nozaki, S., Kawasaki, H., Mizuseki, K., Watanabe, Y., and Sasai, Y.(2005), Directed differentiation of telencephalic precursors fromembryonic stem cells. Nature Neurosci. 8, 288-296; and pamphlet forWO2005/123902]. This method enables efficient differentiation inductionof nerve cells and sensory cells of the forebrain, particularly of thecerebrum and the neural retina. The present inventors also succeeded indifferentiation induction of brainstem tissues such as the cerebellum byadding a growth factor such as Wnt to the medium while performing theSFEB method.

However, an analytical study with mouse embryonic stem cells revealedthat when the SFEB method was applied, about 30% of the cellsdifferentiated into cerebral nerve cells, but the remaining majorityoccurred as a mixture of other kinds of nerve cells. Additionally,cerebral cortex cells accounted for only about 40% of thedifferentiation induction of cerebral nerve cells; the inductionefficiency was not so high. Furthermore, most of the cerebral tissueinduced by a conventional method such as the SFEB method failed to havea clear morphology of cortical tissue, only forming a disarrayed cellmass. Additionally, the conventional SFEB method does not enableefficient differentiation induction of diencephalon tissue, whichdevelops on the most rostral side of the central nervous system.

CITATION LIST Patent Literature

-   patent document 1: WO2005/123902-   patent document 2: JP-A-2008-99662

Non Patent Literature

-   non-patent document 1: Watanabe, K., Ueno, M., Kamiya, D.,    Nishiyama, A., Matsumura, M., Wataya, T., Takahashi, J. B.,    Nishikawa, S., Nishikawa, S.-i., Muguruma, K. and Sasai, Y. (2007) A    ROCK inhibitor permits survival of dissociated human embryonic stem    cells. Nature Biotechnology 25, 681-686-   non-patent document 2: Su, H.-L., Muguruma, K., Kengaku, M.,    Matsuo-Takasaki, M., Watanabe, K., and Sasai, Y. (2006) Generation    of Cerebellar Neuron Precursors from Embryonic Stem Cells.    Developmental Biology 290, 287-296-   non-patent document 3: Ikeda, H., Watanabe, K., Mizuseki, K.,    Haraguchi, T., Miyoshi, H., Kamiya, D., Honda, Y., Sasai, N.,    Yoshimura, N., Takahashi, M. and Sasai, Y. (2005) Generation of    Rx+/Pax6+ neural retinal precursors from embryonic stem cells. Proc.    Natl. Acad. Sci. USA 102, 11331-11336-   non-patent document 4: Hirami Y, Osakada F, Takahashi K, Okita K,    Yamanaka S, Ikeda H, Yoshimura N, Takahashi M. (2009) Generation of    retinal cells from mouse and human induced pluripotent stem cells.    Neurosci Lett. 458(3): 126-31-   non-patent document 5: Watanabe, K., Kamiya, D., Nishiyama, A.,    Katayama, T., Nozaki, S., Kawasaki, H., Mizuseki, K., Watanabe, Y.,    and Sasai, Y. (2005) Directed differentiation of telencephalic    precursors from embryonic stem cells. Nature Neurosci. 8, 288-296

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

It is an object of the present invention to develop a highly practicalmethod that enables differentiation induction of stem cells such as EScells, particularly selective differentiation induction to cells thatform retinal tissue.

Means of Solving the Problems

To explain the low efficiency of differentiation induction of nervecells by the SFEB method, the present inventors hypothesized that theformation of an epithelial structure known as nerve epithelium betweennerve progenitor cells in nerve tissue is necessary for their efficientdifferentiation, proliferation and histologic genesis into various nervecells, including retinal cells, so that the stable formation of theepithelial structure is essential to efficient in vitro production ofnerve cells and central nervous system tissues containing the samerequire. Based on this hypothesis, the present inventors extensivelyinvestigated differentiation induction of embryonic stem cells in theabsence of serum, and found that by forming homogenous aggregates ofstem cells in a serum-free medium, and suspension-culturing theaggregates in the presence of a basement membrane reference standard,nerve cells, particularly retinal progenitor cells, can bedifferentiation-induced from ES cells with high efficiency.

Later, the present inventors found that the retinal progenitor cellsform an optic cup-like structure, and that by culturing them in an organculture broth, a retinal tissue having a functional laminar structurecomparable to the retinal structure after birth can be produced invitro.

The present inventors conducted further investigations based on thesefindings, and have developed the present invention. Accordingly, thepresent invention provides:

[1] a method of differentiation induction of a stem cell into a nerveprogenitor cell, comprising:the step (1) of forming homogenous aggregates of stem cells in aserum-free medium; andthe step (2) of suspension-culturing the homogenous aggregates of stemcells in the presence of a basement membrane reference standard;[2] the method according to [1], wherein the nerve progenitor cell is aretinal progenitor cell;[3] the method according to [2], wherein the basement membrane referencestandard contains an extracellular matrix molecule selected from amonglaminin, type IV collagen, heparan sulfate proteoglycan and entactin;[4] the method according to [2] or [3], wherein the suspension cultureis performed in the presence of KSR;[5] the method according to [4], which is performed in the furtherpresence of Nodal or Activin;[6] a method of morphologically separating or identifying a mass ofretinal progenitor cells, comprising:the step (1) of forming homogenous aggregates of stem cells in aserum-free medium;the step (2′) of suspension-culturing the homogenous aggregates of stemcells in the presence of a basement membrane reference standard to allowan optic cup-like tissue to be self-formed in the aggregate;[7] the method according to [6], wherein the basement membrane referencestandard contains an extracellular matrix molecule selected from amonglaminin, type IV collagen, heparan sulfate proteoglycan and entactin;[8] the method according to [6] or [7], wherein the suspension cultureis performed in the presence of KSR;[9] the method according to [8], which is performed in the furtherpresence of Nodal or Activin;[10] a method of differentiation induction of a retinal layer-specificneuron, comprising:the step (1) of forming homogenous aggregates of stem cells in aserum-free medium;the step (2′) of suspension-culturing the homogenous aggregates of stemcells in the presence of a basement membrane reference standard to allowan optic cup-like tissue to be self-formed in the aggregate; andthe step (3) of suspension-culturing the self-formed optic cup-liketissue in an organ culture broth;[11] the method according to [10], wherein the retinal layer-specificneuron is selected from among photoreceptor cells, horizontal cells,bipolar cells, amacrine cells and ganglion cells;[12] the method according to [10] or [11], wherein the basement membranereference standard contains an extracellular matrix molecule selectedfrom among laminin, type IV collagen, heparan sulfate proteoglycan andentactin;[13] the method according to any one of [10] to [12], wherein thesuspension culture is performed in the presence of KSR;[14] the method according to [13], which is performed in the furtherpresence of Nodal or Activin;[15] a method of producing a retinal tissue in vitro, comprising:the step (1) of forming homogenous aggregates of stem cells in aserum-free medium;the step (2′) of suspension-culturing the homogenous aggregates of stemcells in the presence of a basement membrane reference standard to allowan optic cup-like tissue to be self-formed in the aggregate; andthe step (3) of suspension-culturing the self-formed optic cup-liketissue in an organ culture broth;[16] the method according to [15], wherein the basement membranereference standard contains an extracellular matrix molecule selectedfrom among laminin, type IV collagen, heparan sulfate proteoglycan andentactin;[17] the method according to [15] or [16], wherein the suspensionculture is performed in the presence of KSR;[18] the method according to [17], which is performed in the furtherpresence of Nodal or Activin;[19] a method of producing a retinal layer-specific neuron, comprising:the step (1) of forming homogenous aggregates of stem cells in aserum-free medium;the step (2′) of suspension-culturing the homogenous aggregates of stemcells in the presence of a basement membrane reference standard to allowan optic cup-like tissue to be self-formed in the aggregate; andthe step (3) of suspension-culturing the self-formed optic cup-liketissue in an organ culture broth;[20] the method according to [19], wherein the retinal layer-specificneuron is selected from among photoreceptor cells, horizontal cells,bipolar cells, amacrine cells and retinal ganglion cells;[21] the method according to [19] or [20], wherein the basement membranereference standard contains an extracellular matrix molecule selectedfrom among laminin, type IV collagen, heparan sulfate proteoglycan andentactin;[22] the method according to any one of [19] to [21], wherein thesuspension culture is performed in the presence of KSR;[23] the method according to [22], which is performed in the furtherpresence of Nodal or Activin;[24] a culture product produced by the method according to any one of[1] to [23];[25] a screening method for a test substance, comprising using theculture product according to [24];[26] a toxicity study method for a test substance, comprising using theculture product according to [24];[27] a retina for transplantation containing the culture productaccording to [24].

Effect of the Invention

According to the present invention, it is possible to induce thedifferentiation of a stem cell into a nerve progenitor cell,particularly into a retinal progenitor cell, efficiently. The method ofthe present invention also enables efficient differentiation inductioninto nervous system cells, particularly into retinal cells, a task thathas been difficult to achieve by the conventional method ofdifferentiation induction. Therefore, the method of the presentinvention is particularly useful in applying cytotherapy for diseasesassociated with abnormalities in a nerve tissue, particularly in retinaltissue.

According to the method of the present invention, it is also possible toselectively inducing the differentiation of retinal layer-specificneurons. The retinal tissue obtained by the method of the presentinvention has a laminar structure that is extremely similar to theliving retina. Furthermore, the three-dimensional laminar structure ofthis retinal tissue has formed a functional nerve network that is highlysimilar to the living retina. Therefore, the method of the presentinvention is also highly useful in providing “tissue materials” for usein regenerative medicine for nerve tissues, particularly for retinaltissue, and in providing “tissue materials” that serve well in drugdiscovery seeds screening or toxicity tests of pharmaceuticals, reagentsand the like that act on the retina, and the like, in the production ofpharmaceuticals for nerve tissue disorders, particularly for retinaltissue disorders.

The present invention is also useful in that differentiation of stemcells can be induced without using an animal-derived cell as aninductor, so that the risk levels in the transplantation of cellsobtained by stem cell culture can be reduced to that inallotransplantation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates the steps of the sFEBq method. FIGS. 1B-1O show thataggregates of mouse ES cells obtained by the SFEBq method differentiateinto homogenous nerve cells having an epithelium-like structure.

FIGS. 2A and 2C-2F show that aggregates of mouse ES cells obtained bythe SFEBq method differentiate into cerebral cortex-specific neurons viacerebral cortex progenitor cells. FIG. 2B is a graph showing thepercentage of cells positive for cerebral cortex-specific marker Emx1and positive for the glutamatergic neuron (abundantly present incerebral cortex) marker VGluT1.

FIG. 3 shows that a plurality of optic cup-like tissues are self-formedin an aggregate of mouse ES cells obtained by the modified SFEBq method.

FIG. 4 is a schematic diagram of the living retina.

FIG. 5 shows that a retinal tissue is self-formed bysuspension-culturing in an organ culture broth a mouse optic cup-liketissue self-formed by the modified SFEBq method.

FIG. 6 shows a human optic cup-like tissue self-formed by the modifiedSFEBq method.

FIG. 7 shows a human optic cup-like tissue self-formed by the modifiedSFEBq method in the presence of purified laminin and entactin, andNodal/Activin.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is hereinafter described in detail.

(1) Stem Cells

“A stem cell” refers to a cell capable of retaining a constant potentialfor differentiation even after cell division. Examples of stem cellsinclude embryonic stem cells (ES cells) with pluripotency derived from afertilized egg or clone embryo, somatic stem cells and pluripotent stemcells that are present in tissues in a living organism, hepatic stemcells, dermal stem cells, and reproductive stem cells that serve as thebases for respective tissues, pluripotent stem cells derived from areproductive stem cells, pluripotent stem cells obtained by nuclearreprogrammed somatic cells, and the like.

In particular, “a pluripotent stem cell” refers to a stem cellpermitting in vitro culture, and having the potential fordifferentiating into all cells, but the placenta, constituting the body[tissues derived from the three primary germ layers of the embryo(ectoderm, mesoderm, endoderm)] (pluripotency); embryonic stem cells arealso included. “A pluripotent stem cell” is obtained from a fertilizedegg, clone embryo, reproductive stem cell, or stem cell in tissue. Alsoincluded are cells having differentiation pluripotency similar to thatof embryonic stem cells, conferred artificially by transferring severaldifferent genes to a somatic cell (also referred to as inducedpluripotent stem cells). Pluripotent stem cells can be prepared by amethod known per se. Available methods include, for example, methodsdescribed in Cell 131(5), pp. 861-872, Cell 126(4), pp. 663-676 andelsewhere.

As stem cells, cells derived from a warm-blooded animal, for example,preferably from a mammal, can be used. Mammals include, for example,laboratory animals, including rodents such as mice, rats, hamsters andguinea pigs, and rabbits; domestic animals such as pigs, cattle, goat,horses, and sheep; companion animals such as dogs and cats; primatessuch as humans, monkeys, orangutans, and chimpanzees.

Examples of stem cells useful in the method of the present inventioninclude embryonic stem cells of a mammal or the like established byculturing a pre-implantation early embryo (hereinafter, abbreviated as“embryonic stem cells I”), embryonic stem cells established by culturingan early embryo prepared by nuclear-transplanting the nucleus of asomatic cell (hereinafter, abbreviated as “embryonic stem cells II”),induced pluripotent stem cells (iPS cells) established by transferringseveral different transcriptional factors to a somatic cell, andpluripotent stem cells prepared by modifying a gene on a chromosome ofembryonic stem cells I, embryonic stem cells II or iPS cells using agene engineering technique (hereinafter, abbreviated as “modifiedpluripotent stem cells”).

More specifically, embryonic stem cells I include embryonic stem cellsestablished from an inner cell mass that constitutes an early embryo, EGcells established from a primordial germ cell, cells isolated from acell population possessing the pluripotency of pre-implantation earlyembryos (for example, primordial ectoderm), and cells obtained byculturing these cells.

Embryonic stem cells I can be prepared by culturing a pre-implantationearly embryo according to a method described in the literature(Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, ColdSpring Harbor Laboratory Press (1994)).

Embryonic stem cells II can be prepared using methods reported by Wilmutet al. [Nature, 385, 810 (1997)], Cibelli et al. [Science, 280, 1256(1998)], Akira Iritani et al. [Protein, Nucleic Acid and Enzyme, 44, 892(1999)], Baguisi et al. [Nature Biotechnology, 17, 456 (1999)], Wakayamaet al. [Nature, 394, 369 (1998); Nature Genetics, 22, 127 (1999); Proc.Natl. Acad. Sci. USA, 96, 14984 (1999)], Rideout III et al. [NatureGenetics, 24, 109 (2000)] and others, for example, as described below.

By extracting the nucleus of a mammalian cell and then reprogramming thenucleus (an operation to return the nucleus to a state to resumedevelopment), initiating development using a method involving injectioninto an enucleated unfertilized egg of a mammal, and culturing the eggthat has started development, an egg that has the nucleus of anothersomatic cell, and has begun normal development, is obtained.

A plurality of methods of reprogramming the nucleus of a somatic cellare known. For example, the nucleus can be reprogrammed by changing themedium used to culture the nucleus donor cell from a medium containing 5to 30%, (preferably 10%) of fetal calf serum (e.g., M2 medium) to anoligotrophic medium containing 0 to 1% (preferably 0.5%) of fetal calfserum, and culturing the cell for 3 to 10 days (preferably 5 days) toinduce the cell cycle into a resting phase state (G0 stage or G1 stage).

The nucleus can also be reprogrammed by injecting the nucleus of thenucleus donor cell into an enucleated unfertilized egg of a mammal ofthe same species, and culturing the cell for several hours, preferablyfor about 1 to 6 hours.

The reprogrammed nucleus is able to begin development in the enucleatedunfertilized egg. A plurality of methods of allowing the reprogrammednucleus to begin development in the enucleated unfertilized egg areknown. By transplanting a nucleus reprogrammed by inducing the cellcycle to a resting phase state (phase G0 or phase G1) into an enucleatedunfertilized egg of a mammal of the same species by the electrofusionmethod and the like, the egg can be activated and allowed to begindevelopment.

A nucleus reprogrammed by injecting the nucleus into an enucleatedunfertilized egg of a mammal of the same species is transplanted back toan enucleated unfertilized egg of a mammal of the same species by amethod using a micromanipulator or the like, and stimulated with an eggactivator (e.g., strontium and the like), and thereafter treated with aninhibitor of cell division (e.g., cytochalasin B and the like) tosuppress the release of the second polar body, whereby development canbe initiated. This method is suitable when the mammal is, for example, amouse or the like.

Provided that an egg that once began to develop is obtained, embryonicstem cells can be acquired using publicly known methods described inManipulating the Mouse Embryo A Laboratory Manual, Second Edition, ColdSpring Harbor Laboratory Press (1994); Gene Targeting, A PracticalApproach, IRL Press at Oxford University Press (1993); Biomanual Series8 Gene Targeting, Preparation of Mutant Mice Using ES Cells, Yodosha(1995) and the like.

iPS cells can be produced by transferring Oct3/4, Sox2 and Klf4 (c-Mycor n-Myc further added as required) to somatic cells (e.g., fibroblasts,dermal cells and the like) (Cell, 126: p. 663-676, 2006; Nature, 448: p.313-317, 2007; Nat Biotechnol, 26: p. 101-106, 2008; Cell 131: 861-872,2007).

Modified pluripotent stem cells can be prepared by, for example,homologous recombination technology. Examples of the gene on thechromosome to be modified in preparing modified pluripotent stem cells,histocompatibility antigen genes, genes related to diseases based onneural cell disorders, and the like. A modification of the target geneon the chromosome can be performed using methods described inManipulating the Mouse Embryo A Laboratory Manual, Second Edition, ColdSpring Harbor Laboratory Press (1994); Gene Targeting, A PracticalApproach, IRL Press at Oxford University Press (1993); Biomanual Series8 Gene Targeting, Preparation of Mutant Mice Using ES Cells, Yodosha(1995) and the like.

Specifically, for example, a genomic gene of a target gene to bemodified (for example, histocompatibility antigen genes, disease-relatedgenes and the like) is isolated, and a target vector for homologousrecombination of the target gene is prepared using the genomic geneisolated. By transferring the target vector prepared to an embryonicstem cell, and selecting cells undergoing homologous recombinationbetween the target gene and the target vector, stem cells having amodified gene on the chromosome can be prepared.

Methods of isolating a genomic gene of a target gene include publiclyknown methods described in Molecular Cloning, A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press (1989), CurrentProtocols in Molecular Biology, John Wiley & Sons (1987-1997) andelsewhere. A genomic gene of a target gene can also be isolated by usinga genomic DNA library screening system (produced by Genome Systems),Universal GenomeWalker™ Kits (produced by CLONTECH) and the like.

Preparation of a target vector for homologous recombination of a targetgene and efficient sorting of a homologous recombinant can be achievedby a method described in Gene Targeting, A Practical Approach, IRL Pressat Oxford University Press (1993); Biomanual Series 8 Gene Targeting,Preparation of Mutant Mice Using ES Cells, Yodosha (1995) and elsewhere.The target vector used may be any one of the replacement type and theinsertion type. Useful methods of sorting include positive selection,promoter selection, negative selection, poly A selection and the like.

Available methods of selecting a desired homologous recombinant fromamong the sorted cell lines include Southern hybridization, PCR and thelike for genomic DNA.

Stem cells are available from specified organizations, and commercialproducts may be purchased. For example, the human embryonic stem cellsKhES-1, KhES-2 and KhES-3 are available from the Institute for FrontierMedical Sciences, Kyoto University. Examples of mouse embryonic stemcells include EB5 cells and the like.

Stem cells can be cultured for maintenance by a method known per se. Forexample, stem cells can be maintained by cultivation without feedercells with the addition of fetal calf serum (FCS), Knockout™ SerumReplacement (KSR), and LIF.

(2) Cells Permitting Differentiation Induction by the Method of thePresent Invention

According to the present invention, differentiated cells can be obtainedfrom stem cells, preferably pluripotent stem cells such as embryonicstem cells. The cells differentiation-induced from a stem cell by themethod of the present invention are preferably nervous system cells. Thecells are more preferably nerve stem cells, particularly preferablynerve progenitor cells, most preferably retinal progenitor cells. Nervecells obtained via nerve progenitor cells can also be obtained by thepresent invention. While the type of such nerve cells is notparticularly limited, retinal cells are preferable. The cells obtainedby the method of the present invention can be identified by a methodknown per se, for example, by the expression of a cell marker.

Examples of markers of nervous system cells include, but are not limitedto, Rx, NCAM, TuJ1, tyrosine hydroxylase (TH), serotonin, nestin, MAP2,MAP2ab, NeuN, GABA, glutamates, ChAT, Sox1, Bf1, Emx1, VGluT1, Pax, Nkx,Gsh, Telencephalin, GluR1, CamKII, Ctip2, Tbr1, Reelin, Tbr1, Brn2 andthe like. The expression of a marker gene is analyzed by, for example,performing quantitative PCR using the 7500 Fast Real-Time PCR System(Applied Biosystems) in accordance with the manufacturer's instructions,and normalizing the obtained data by the expression of GAPDH. The methodof quantitative PCR is obvious to those skilled in the art.Alternatively, cells may be manipulated to allow the desired marker geneto be expressed as a fusion protein of a marker gene product and GFP orthe like (knock-in). It is also possible to detect the expression of theprotein using an antibody specific for a marker gene product.Hereinafter, to exemplify cells that can be differentiation-induced bythe method of the present invention, nerve stem cells are described indetail.

A nerve stem cell refers to a cell having both the potential fordifferentiation into nerve cells, astrocytes and oligodendrocytes andthe potential for autoreproduction, functioning in the brain to supplynerve cells, astrocytes and oligodendrocytes. The nerve stem cells thatdifferentiate into nerve cells, in particular, are called nerveprogenitor cells. In the present specification, nerve stem cells areunderstood to include nerve progenitor cells.

Available methods of confirming the identity of the cell obtained as anerve stem cell include a method wherein the cell is actuallytransplanted to a living brain and its differentiating potential isconfirmed, a method wherein the nerve stem cell isdifferentiation-induced to nerve cells/astrocytes/oligodendrocytes invitro, and the like [Mol. Cell. Neuroscience, 8, 389(1997); Science,283, 534(1999)]. Nerve stem cells having these functions are stainablewith an anti-nestin antibody that recognizes cytoskeletal proteinnestin, which is a marker whose expression has been confirmed in nerveprogenitor cells, and an anti-Sox1 antibody that recognizes the nuclearfactor Sox1 [Science, 276, 66(1997)]. Therefore, it is also possible toconfirm the identity of the nerve stem cell by staining with ananti-nestin antibody or anti-Sox1 antibody. However, despite theiridentity as nerve progenitor cells, retinal progenitor cells areexceptionally not stainable with an anti-nestin antibody or anti-Sox1antibody, but are instead stainable with anti-Rx and anti-Pax6antibodies, which recognize Rx and Pax6, which are nuclear factorsexpressed in retinal progenitor cells. Therefore, retinal progenitorcells can be identified as cells that are positive for Rx and Pax6 andnegative for nestin and Sox1 (Ikeda et al, PNAS 2005).

Nerve progenitor cells are not particularly limited, as far as they havethe potential for differentiating into various nerve cells, and theyinclude cerebral progenitor cells, cerebellar progenitor cells, midbrainprogenitor cells, after-brain progenitor cells, diencephalon progenitorcells, retinal progenitor cells and the like. In the present invention,preferred nerve progenitor cells are diencephalon progenitor cells andretinal progenitor cells, with greater preference given to retinalprogenitor cells. The method of the present invention allows theseoptionally chosen nerve progenitor cells to be differentiation-induced;in particular, diencephalon progenitor cells and retinal progenitorcells, preferably retinal progenitor cells, can bedifferentiation-induced efficiently.

Alternatively, nerve progenitor cells, particularly retinal progenitorcells, obtained by the method of the present invention can becharacterized by cell markers. Nerve progenitor cell markers include,but are not limited to, Rx, NCAM, Sox1, Bf1, nestin, Emx1, Pax6, Nkx2.1,and Gsh2. Retinal progenitor cell markers, in particular, include Rx,Pax6, Chx10 (with the provision of co-expression with Ki67) and thelike.

Nerve cell markers include, but are not limited to, TuJ1, tyrosinehydroxylase (TH), serotonin, MAP2, MAP2ab, NeuN, GABA, glutamates, ChAT,VGluT1, GluR1, CamKII, Reelin, Telencephalin, Ctip2, Tbr1, Tbr2, Brn2,L7 and the like. The nerve progenitor cells obtained by the method ofthe present invention are Rx-positive at a high frequency of at least60% or more, preferably 80% or more, more preferably about 80 to 90%.

According to the present invention, it is also possible to inducedifferentiation of stem cell into retinal cells via retinal progenitorcells. Particularly, the retinal cells obtained by differentiationinduction by the present invention are obtained as a constituent of acell aggregate having a three-dimensional laminar structure that isextremely similar to the living retina. Hence, the retinal cells of thepresent invention can assume a three-dimensional laminar structure thatis morphologically extremely similar to the living retina, so thatneurons specific for respective retinal layers (herein, these aredescribed as “retinal layer-specific neurons” together) are included inthe scope of the invention.

The retinal cells obtained by the present invention include, but are notlimited to, all the cells that constitute the retina; cells constitutingthe individual retinal layers (retinal layer-specific neurons) include,for example, photoreceptor cells, horizontal cells, bipolar cells,amacrine cells, retinal ganglion cells and the like. According to thepresent invention, these cells can be efficiently obtained bydifferentiation induction from a stem cell. The type of the retinalcells obtained by the present invention can be identified by a methodknown per se, for example, by the expression of a cell marker.

Retinal cell markers include, but are not limited to, Rx (retinalprogenitor cells), PAX6 (progenitor cells), nestin (expressed inhypothalamic neuron progenitor cells, but not in retinal progenitorcells), Sox1 (expressed in hypothalamic nerve epithelium, but not in theretina), Crx (photoreceptor cell progenitor cells) and the like.Particularly, markers of the above-described retinal layer-specificneurons include, but are not limited to, Chx10 (bipolar cells), L7(bipolar cells), Tuj1 (ganglion cells), Brn3 (ganglion cells),calretinin (amacrine cells), calbindin (horizontal cells), rhodopsin(photoreceptor cells), recoverin (photoreceptor cells), RPE65 (pigmentepithelial cells), Mitf (pigment epithelial cells) and the like.

(3) Method of Differentiation Induction of the Present Invention

The present invention provides a method of differentiation induction ofa stem cell into nerve progenitor cells, comprising the step of forminghomogenous aggregates of stem cells in a serum-free medium and the stepof suspension-culturing the homogenous aggregates of stem cells in thepresence of a basement membrane reference standard.

(3-1) The Step of Forming Homogenous Aggregates of Stem Cells in aSerum-Free Medium [Step (1)]

“Forming homogenous aggregates of stem cells” refers to formingqualitatively homogenous aggregates of stem cells by allowing “a givennumber of dispersed stem cells to aggregate quickly” in allowing stemcells to assemble and form aggregates of stem cells and culturing theaggregates (aggregate culture), referring particularly to promoting theepithelization of cells deriving from stem cells by allowing “the cellsto aggregate quickly”. Hence, as used herein, the term “to allow thecells to aggregate quickly” refers to forming with high reproducibilityan epithelium-like structure in the cells produced by allowing stemcells to aggregate homogenously.

Any method may be employed to form homogenous aggregates of stem cells,as far as homogenous aggregates of stem cells are formed by allowing“the cells to aggregate quickly”, and an epithelium-like structure ofthe cells produced from the stem cells is formed with highreproducibility. Such methods include, for example, a method whereincells are enclosed in small spaces using a plate with small wells(96-well plate), micropores or the like, a method wherein cells areaggregated by centrifugation for a short time using small centrifugaltubes, and the like.

Any incubator can be used to form aggregates, as far as it allowshomogenous aggregates of stem cells to be formed by allowing “the cellsto aggregate quickly”; those skilled in the art are able to determinethe choice as appropriate. Such incubators include, for example, flasks,tissue culture flasks, dishes, Petri dishes, tissue culture dishes,multi-dishes, microplates, micro-well plates, micropores, multi-plates,multi-well plates, chamber slides, Petri dishes, tubes, trays, culturingbags, and roller bottles. From the viewpoint of forming homogenousaggregates, it is preferable that these incubators be non-cell-adhesive.Useful non-cell-adhesive incubators include incubators whose surfaceshave not undergone an artificial treatment (e.g., coating withextracellular matrix and the like) for improving the cell adhesiveness.

A medium used to form aggregates can be prepared using a medium in usefor animal cell culture as a basal medium. Any basal medium availablefor culturing animal cells can be used; examples include, but are notlimited to, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEMmedium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium,Eagle's MEM medium, αMEM medium, DMEM medium, Ham's medium, RPMI 1640medium, Fischer's medium, a mixed medium thereof and the like.

Here, a serum-free medium used to form aggregates means a medium notcontaining an unadjusted or unpurified serum. Any such serum-free mediumcan be used in the present invention. However, to avoid thepainstakingness in preparing the serum-free medium, a serum-free medium(GMEM or dMEM, 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential aminoacids Mix, 1 mM sodium pyruvate) supplemented with an appropriate amount(e.g., 1-20%) of commercially available KSR can be used.

The serum-free medium may contain a serum substitute. The serumsubstitute can be, for example, one containing as appropriate albumin,transferrin, fatty acids, collagen precursor, trace elements,2-mercaptoethanol or 3′-thiolglycerol, or equivalents thereof and thelike. These serum substitutes can be prepared by, for example, a methoddescribed in WO98/30679. Also, to carry out the method of the presentinvention more conveniently, a commercially available serum substitutecan be utilized. Examples of such commercially available serumsubstitutes include Chemically-defined Lipid Concentrated (produced byGibco Company) and Glutamax (produced by Gibco Company).

The serum-free medium used for the suspension culture can also containfatty acids or lipids, amino acids (e.g., non-essential amino acids),vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol,pyruvic acid, buffering agents, inorganic salts and the like.

The concentration of stem cells at the time of aggregate formation canbe set as appropriate by those skilled in the art, such that aggregatesof stem cells will be formed more homogenously and efficiently. Theconcentration of stem cells at the time of aggregate formation may beany concentration that allows homogenous aggregates of stem cells to beformed. In case of differentiation culture of mouse ES cells using a96-well microwell plate, for example, their suspensions prepared toobtain a cell density of about 1×10³ to about 5×10³ cells, preferablyabout 2×10³ to about 4×10³ cells, per well, are added to the plate, andthe plate is kept to stand to allow aggregates to be formed. In case ofhuman ES cells, suspensions prepared to obtain a cell density of about1×10³ to about 12×10³ cells, preferably about 4×10³ to about 10×10³cells, per well, are used.

Other culturing conditions such as culturing temperature and CO₂concentration at the time of aggregate formation can be set asappropriate. The culturing temperature is not particularly limited, andis, for example, about 30 to 40° C., preferably about 37° C. The CO₂concentration is, for example, about 1 to 10%, preferably about 5%.

Although the time to the formation of aggregates can be determined asappropriate according to the choice of stem cell used, as far as cellsare allowed to aggregate quickly, it is desirable that the formation beperformed as soon as possible to ensure the formation of homogenousaggregates. Conventionally, this formation of aggregates is performedover about 2 days (see, for example, Watanabe, K. et al., NatureNeurosci. 8, 288-296, Schuldiner M, Benvenisty N. Factors controllinghuman embryonic stem cell differentiation. Methods Enzymol. 2003; 365:446-461). In the present invention, by contrast, this time is shortenedto enable efficient differentiation induction of desired nerve cells andthe like. In case of mouse embryonic stem cells, for example, it isdesirable that aggregates be formed preferably within 12 hours, morepreferably within 6 hours. Meanwhile, in case of human embryonic stemcells, it is desirable that aggregates be formed preferably within 24hours, more preferably within 12 hours. If this time is exceeded,homogenous aggregates of stem cells cannot be formed, which in turn cancause a remarkable reduction in differentiation efficiency in thesubsequent step. This time to aggregate formation can be adjusted asappropriate by choosing a tool for cell aggregation, centrifugalconditions and the like by those skilled in the art.

Those skilled in the art are able to make a judgement concerning the“homogenous” formation of aggregates of stem cells and the formation ofan epithelium-like structure in each cell type that forms aggregates, onthe basis of the size of the aggregate masses and the number of cellstherein, macroscopic morphology, microscopic morphology as analyzed byhistological staining and uniformity thereof, the expression ofdifferentiation and non-differentiation markers and uniformity thereof,the control of the expression of differentiation markers andsynchronicity thereof, inter-aggregate reproducibility ofdifferentiation efficiency, and the like.

Specifically, homogenous aggregates of stem cells can be formed by, forexample, a method wherein embryonic stem cells are cultured formaintenance, followed by dispersion treatment, and suspended in anappropriate medium (e.g., Glasgow MEM medium supplemented with 10% KSR,0.1 mM non-essential amino acid solution, 2 mM glutamine, 1 mM pyruvicacid and 0.1 mM 2-mercaptoethanol; may contain appropriate amounts offactors described below, added as required, and the like), and the cellsare suspended in 150 μL of the above-described medium at 3×10³ cells perwell using a non-cell-adhesive U-bottom 96-well culture plate to formaggregates rapidly.

(3-2) The Step of Suspension-Culturing the Homogenous Aggregates of StemCells in a Serum-Free Medium in the Presence of a Basement MembraneReference Standard [Step (2)]

This is a step wherein the homogenous aggregates of stem cells formed inthe step (1) are subjected to suspension culture in the presence of abasement membrane reference standard to induce the differentiation ofstem cells.

“The basement membrane reference standard” may be any one that containsa basement membrane constituent component having the function ofcontrolling the morphology, differentiation, proliferation, motor,functional expression and the like of epithelial cell-like cells whendesired cells capable of forming a basement membrane are seeded andcultured thereon. Such a basement membrane reference standard can beprepared by, for example, removing the cells capable of forming thebasement membrane, adhering to a support via the basement membrane,using a solution capable of dissolving the lipids of the cells, analkali solution and the like.

Preferred basement membrane reference standards include commerciallyavailable products as basement membrane components (e.g., Matrigel) andthose containing an extracellular matrix molecule publicly known as abasement membrane component (e.g., laminin, type IV collagen, heparansulfate proteoglycan, entactin and the like). These extracellular matrixmolecules to be used are desirably purified products.

As such extracellular matrix molecules, laminin and entactin can bepreferably used. Particularly, when Nodal and Activin to be mentionedlater are concurrently used for performing the present invention,purified laminin and entactin can be preferably used as basementmembrane components.

Matrigel is a basement membrane preparation derived from Engelbreth HolmSwarn (EHS) mouse sarcoma. The major components of Matrigel are type IVcollagen, laminin, heparan sulfate proteoglycan, and entactin. Inaddition, TGF-β, fibroblast growth factor (FGF), tissue plasminogenactivator, and the growth factor naturally produced by EHS tumors arealso contained. “Growth factor reduced products” of Matrigel have lowerconcentrations of growth factors than in ordinary Matrigel; the standardconcentrations thereof are <0.5 ng/ml for EGF, <0.2 ng/ml for NGF, <5μg/ml for PDGF, 5 ng/ml for IGF-1, and 1.7 ng/ml for TGF-β. In themethod of the present invention, it is preferable to use “a growthfactor reduced product”.

The concentration of the basement membrane reference standard added tothe medium for suspension culture in this step is not particularlylimited, as far as the epithelial structure of a nerve tissue (e.g.,retinal tissue) is stably maintained. For example, purified laminin andentactin can be preferably used. Particularly, when Nodal and Activin tobe mentioned later are used (laminin/entactin complex is used), it isgenerally added at a concentration of 1 μg/mL-5000 μg/mL, preferably 10μg/mL-2000 μg/mL, more preferably 20 μg/mL-1000 μg/mL, and mostpreferably 50 μg/mL-500 μg/mL, to a medium. When using Martigel, forexample, it is added preferably in a volume 1/100 to 1/20, morepreferably in a volume 1/100 to 1/50, of the volume of the culturebroth. Although the basement membrane reference standard may be added tothe medium already at the start of culturing the stem cell, it is addedto the medium preferably within several days after the start ofsuspension culture (e.g., 1 to 3 days after the start of suspensionculture).

“To suspension-culture homogenous aggregates of stem cells” or “toculture homogenous aggregates of stem cells as suspended aggregates(also referred to as aggregate masses)” refers to culturing thepopulation of stem cells having assembled to form homogenous aggregates,obtained in the above-described step (1), in a culture medium underconditions that are non-adhesive to the cell incubator (herein, theabove-described steps (1) and (2) are sometimes described as “themodified SFEBq method” together; the method wherein no basement membranereference standard is used in the step (2) is described as “the SFEBqmethod”). When stem cells are suspension-cultured, the culture ispreferably performed in the absence of feeder cells to facilitate theformation of suspended aggregates, and/or to achieve efficient inductionof differentiation (e.g., induction of differentiation into ectodermalcells such as nervous system cells).

A medium used in the suspension culture of the aggregates obtained inthe above-mentioned step (1) can be prepared with a medium for animalcell culture as the basal medium. The basal medium is not particularlylimited, as far as it is a medium that can be used for animal cellculture; for example, BME medium, BGJb medium, CMRL 1066 medium, GlasgowMEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199medium, Eagle MEM medium, αMEM medium, DMEM medium, Ham medium, RPMI1640 medium, Fischer's medium, and a mixed medium thereof and the likecan be mentioned. Unless otherwise specified, the medium used in thestep (1) may be used as it is for the suspension culture.

When the above-mentioned aggregates are suspension-cultured, aserum-free medium is used as the medium. Here, a serum-free medium meansa medium that does not contain unadjusted or unpurified serum. A mediumcontaining a purified blood-derived component or animal tissue-derivedcomponent (for example, growth factor) is to be construed as aserum-free medium, as far as it does not contain unadjusted orunpurified serum.

The serum-free medium used in the suspension culture can be, forexample, one containing a serum substitute. The serum substitute can,for example, be one containing as appropriate an albumin (for example,lipid-rich albumin), transferrin, fatty acids, insulin, collagenprecursor, trace elements, 2-mercaptoethanol or 3′-thiolglycerol, ortheir equivalents and the like. Such serum substitutes can be preparedby, for example, a method described in WO98/30679. To facilitate easierimplementation of a method of the present invention, commerciallyavailable serum substitutes can be utilized. Examples of suchcommercially available serum substitutes include Knockout SerumReplacement (KSR), Chemically-defined Lipid Concentrated (produced byGibco) and Glutamax (produced by Gibco).

In addition, the serum-free medium used in the method of the presentinvention can contain fatty acids or lipids, amino acids (for example,non-essential amino acids), vitamins, growth factors, cytokines,anti-oxidants, 2-mercaptoethanol, pyruvic acid, buffering agents,inorganic salts and the like. For example, 2-mercaptoethanol can be usedwithout particular limitations as far as it is used at a concentrationsuitable for stem cell culture, and it can be used at concentrations of,for example, about 0.05 to 1.0 mM, preferably about 0.1 to 0.5 mM, morepreferably about 0.2 mM.

The serum-free medium used for the suspension culture is notparticularly limited, as far as it is as described above. However, fromthe viewpoint of avoiding the painstakingness in the preparation, and ofefficiently inducing the differentiation of stem cells into nerveprogenitor cells, preferably retinal progenitor cells, it is preferablethat a serum-free medium (GMEM or dMEM, 0.1 mM 2-mercaptoethanol, 0.1 mMnon-essential amino acids Mix, 1 mM sodium pyruvate) supplemented withan appropriate amount of commercially available KSR (Knockout SerumReplacement) be used as the serum-free medium. The amount of KSR addedto the serum-free medium is not particularly limited. In case of mouseES cells, for example, the amount added is normally 1 to 20% (v/v). Whenretinal progenitor cells are to be differentiation-induced from mouse EScells more efficiently, the amount added is preferably 1 to 5%, mostpreferably 2%. In case of human ES cells, the amount of KSR added isnormally 1 to 20%. When retinal progenitor cells are to bedifferentiation-induced from human ES cells more efficiently, the amountof KSR added is preferably 2 to 20%. By suspension-culturing theaggregates with the addition of KSR, in addition to the aforementionedbasement membrane reference standard, the method of differentiationinduction of the present invention described below and the like can beperformed more efficiently.

The culture vessel used for the suspension culture is not particularlylimited, as far as it allows suspension culture of cells; examplesinclude flasks, tissue culture flasks, dishes, Petri dishes, tissueculture dishes, multi-dishes, microplates, micro-well plates,multi-plates, multi-well plates, chamber slides, Petri dishes, tubes,trays, culturing bags, and roller bottles.

When aggregates are suspension-cultured, the culture vessel ispreferably non-adhesive to cells. As the non-adhesive-to-cell culturevessel, a culture vessel whose surface has not been artificially treatedfor the purpose of increasing the adhesiveness to cells (e.g., coatingtreatment with extracellular matrix and the like) can be used.

Other culturing conditions such as culturing temperature, CO₂concentration and O₂ concentration at the time of aggregate suspensionculture can be set as appropriate. The culturing temperature is notparticularly limited, and is, for example, about 30 to 40° C.,preferably about 37° C. The CO₂ concentration is, for example, about 1to 10%, preferably about 5%. Meanwhile, when retinal progenitor cellsare to be differentiation-induced, and taking into account the highoxygen demand thereby, the O₂ concentration is, for example, 20 to 70%,preferably 20 to 50%, more preferably 20 to 40%. The culturing time inthis step is not particularly limited, and is normally 48 hours or more,preferably 7 days or more.

After suspension culture, the aggregates may be kept as they are ordispersion-treated (e.g., trypsin/EDTA treatment), and the cells may befurther cultured under adhesive conditions (hereinafter, described as“adhesion culture” if required). If adhesion culture is performed, it ispreferable that a cell-adhesive incubator, for example, an incubatorcoated with an extracellular matrix and the like (e.g., poly-D-lysine,laminin, entactin, fibronectin) be used. Culturing conditions such asculturing temperature, CO₂ concentration, O₂ concentration and culturingtime in adhesion culture can easily be determined by those skilled inthe art.

In suspension culture and adhesion culture, a known differentiationinducer can be used in combination. For example, when nerve progenitorcells are to be differentiation-induced from an embryonic stem cell, aknown inducer of differentiation into nerve progenitor cells can be usedin combination. Examples of such differentiation inducers include NGF[Biochem. Biophys. Res. Commun., 199, 552(1994)], retinoic acid [Dev.Biol., 168, 342(1995); J. Neurosci., 16, 1056(1996)], BMP inhibitoryfactor [Nature, 376, 333-336 (1995)], IGF [Genes & Development, 15,3023-8 (2003)], Nodal inhibitor, Wnt inhibitor [Nature Neurosci. 8,288-296 (2005)], Activin (Proc Natl. Acad. Sci. USA, 2005 Aug. 9;102(32) 11331-6), and the like.

In the present invention, when laminin and entactin are used as abasement membrane reference standard, Nodal and Activin are desirablyused as known differentiation inducers.

The amount of the differentiation inducer to be used in combination isnot particularly limited and those of ordinary skill in the art canprepare a suitable amount that induces a desired differentiation. WhenNodal and Activin are used for differentiation induction of retinalprogenitor cells, Nodal is generally added to a culture solution at aconcentration of 50 ng/mL-4000 ng/mL, preferably 200 ng/mL-2000 ng/mL,and Activin is generally added to a culture solution at a concentrationof 20 ng/mL-2000 ng/mL, preferably 50 ng/mL-500 ng/mL. The culturesolution is desirably exchanged daily. By adding a differentiationinducer besides the above-mentioned basement membrane referencestandards when performing a floating culture of aggregates, thedifferentiation induction method to be described below can be performedfurther efficiently.

The timing of addition of a differentiation inducer is not particularlylimited, and it may be added from the initial stage of differentiationinduction or at a suitable time point. When Nodal and Activin are usedfor the above-mentioned object, they may be added one day after thestart of the culture and continuously added for 7 to 10 days thereafter.

According to the above-described suspension culture method andcombination of suspension culture and adhesion culture, nerve progenitorcells can be obtained from a stem cell by setting duration ofcultivation and the like as appropriate. Particularly in the step (2),when homogenous aggregates of stem cells are suspension-cultured in thepresence of a basement membrane reference standard for several days toseveral tens of days (e.g., 7 to 12 days for mouse ES cells, 20 to 40days for human ES cells), self-formation of a plurality of opticcup-like protrusion structures as shown in FIG. 3 (hereinafter, thisstructure is described as “optic cup-like tissue”) is observed in theaggregate of stem cells [hereinafter, of the step (2), particularly thestep of allowing an optic cup-like tissue to be self formed in theaggregate is described as “step (2′)”].

The identity of the retinal progenitor cells obtained by theabove-described suspension culture method or combination of suspensionculture and adhesion culture can be confirmed by the presence or absenceof the expression of a marker gene and the like or the shape and thelike of the cells or tissue as an index, which may be combined asrequired. Choice of marker gene for retinal progenitor cells and how toanalyze their expression are as described in (2) above.

The thus-obtained optic cup-like tissue not only simply morphologicallyhas an optic cup-like protrusion in the aggregate, but also exhibits ahigh level of expression of the retinal progenitor cell marker Rx fromthe cells constituting the tissue. Also observed in the outer part ofthe protrusion is a layer of retinal pigment epithelial cells thatexpress Pax6. This structure of optic cup-like tissue is extremelysimilar to the structure of optic cup tissue in the genesis of a livingorganism. Therefore, according to the method of the present invention,it is possible to produce not only nerve progenitor cells (preferablyretinal progenitor cells), but also a self-assembled optic cup-liketissue, from a stem cell.

Because the optic cup-like tissue obtained is self-formed as aprotrusion from the aggregate, it is possible to obtain a highly puremass of retinal progenitor cells by separating the protrusion.Accordingly, the present invention provides a method of separating oridentifying a mass of retinal progenitor cells, comprising theabove-described steps (1) and (2′).

A mass of retinal progenitor cells can be obtained by cutting out theself-formed optic cup-like tissue from the aggregate physically andmorphologically. Therefore, this method makes it possible to easilyseparate the mass of retinal progenitor cells. Because this methodobviates the operation of confirming the position of retinal progenitorcells using a retinal progenitor cell marker and the like in obtainingthe retinal progenitor cells by suspension-culturing aggregates of stemcells, a mass of retinal progenitor cells can easily be obtained bymerely cutting out the cell mass formed as a protrusion in theaggregate.

(3-3) The Step of Suspension-Culturing the Formed Optic Cup-Like Tissuein Organ Culture Broth [Step (3)]

This is a step wherein the optic cup-like tissue obtained in the step(2′) is suspension-cultured in an organ culture broth.

In this step, the optic cup-like tissue self-formed in the step (2′) issuspension-cultured in an organ culture broth. The optic cup-like tissueused may be the aggregate of stem cells containing the optic cup-liketissue; the optic cup-like tissue formed as a protrusion from theaggregate of stem cells may be cut out physically and morphologically,and this may be suspension-cultured in an organ culture broth. When theoptic cup-like tissue has been cut out, the entire aggregate of stemcells can be handled as a mass of Rx-positive retinal progenitor cells;therefore, it is possible to induce the differentiation of retinalprogenitor cells more efficiently to produce retinal tissue and retinallayer-specific neurons.

The optic cup-like tissue can be cut out using any method; it ispossible to cut out the tissue from an aggregate of stem cells usingmicrotweezers and the like.

While the organ culture broth used to culture the optic cup-like tissueis not particularly limited, an organ culture broth in common use forinduction of retinal cells is preferably used. Examples include (1)DMEM/F12/N2+0.5 μM retinoic acid, (2) 66% E-MEM-HEPES+33% HBSS+1% FCS+N2supplement+5.75 mg/ml glucose+200 mM L-glutamine+20 ng/ml aFGF+20 ng/mlbFGF+20 nM Shh+1 mM retinoic acid+100 mM taurine, or (3) G-MEM+5% KSR+N2supplement+0.1 mM non-essential amino acids+1 mM pyruvate+0.1 mM2-mercaptoethanol+1 mM retinoic acid+100 mM taurine and the like.

The optic cup-like tissue is cultured in an organ culture broth. Theincubator used in the suspension culture of the optic cup-like tissuemay be the same as the incubator used in the above-described step (2).Other culturing conditions such as culturing temperature, CO₂concentration and O₂ concentration at the time of optic cup-like tissuesuspension culture can be set as appropriate. The culture temperature isnot particularly limited, and is, for example, about 30 to 40° C.,preferably about 37° C. The CO₂ concentration is, for example, about 1to 10%, preferably about 5%. Meanwhile, the O₂ concentration is, forexample, 20 to 70%, preferably 20 to 60%, more preferably 30 to 50%,when retinal progenitor cells are to be differentiation-induced, andtaking into account the high oxygen requirement thereby.

The culturing time in this step is not particularly limited, and isnormally 48 hours or more, preferably 7 days or more.

The optic cup-like tissue self-formed in the step (2′) is self-inducedto a retinal tissue through this step. Hence, by suspension-culturingthe optic cup-like tissue self-formed in the step (2′) in an organculture broth, a retinal tissue or a cell population constituting theretinal tissue is self-induced in vitro. Accordingly, the presentinvention provides a method of producing a retinal tissue in vitro byself formation of retinal tissue and a method of producing a cellpopulation constituting the retinal tissue. Here, “in vitro” merelyrefers to being not in a living organism.

As shown in FIG. 4, the retinal tissue in a living organism has astructure wherein nerve cells are orderly arranged in a regularfive-layer laminar structure so as to allow the incidental light throughthe cornea and lens to be received by surface photoreceptor cells andconverted to an electric signal, to transmit information in the order ofbipolar cells and nerve ganglion cells, and to finally transmit thesignal to the cerebrum. The retinal tissue self-formed by the method ofthe present invention is not a mere assembly of retinal cells, butsurprisingly has a structure that is morphologically extremely similarto the living retinal tissue.

The retinal tissue self-formed by the present invention has aself-formed five-laminar structure of nerve cells in regular arrangementin the same order as in the retinal tissue of a living organism. Thefive layers are configured by different types of retinal cells(photoreceptor cells, horizontal cells, bipolar cells, amacrine cells,ganglion cells); by assuming this structure, the retinal tissue iscapable of transmitting light stimuli from outside of the body to thecentral nervous system as electric stimuli.

According to the present invention, a method of selectivedifferentiation induction from a stem cell to a cell populationconstituting this retinal tissue, i.e., “retinal layer-specific neuron”,is provided. Also provided is a method of producing “a retinallayer-specific neuron” from a stem cell.

The retinal layer-specific neuron thus obtained may be kept as it is ordispersion-treated (e.g., trypsin/EDTA treatment), and the cells may befurther cultured under adhesive conditions. In adhesion culture, it ispreferable that a cell-adhesive incubator, for example, an incubatorcoated with an extracellular matrix and the like (e.g., poly-D-lysine,laminin, entactin, fibronectin), be used. Culturing conditions for theadhesion culture, such as culturing temperature, CO₂ concentration, andO₂ concentration, can easily be determined by those skilled in the art.In this operation, the cells may be cultured in the presence of a knowndifferentiation inducer. Examples of such differentiation inducersinclude NGF [Biochem. Biophys. Res. Commun., 199, 552(1994)], retinoicacid [Dev. Biol., 168, 342 (1995); J. Neurosci., 16, 1056(1996)], BMPinhibitory factor [Nature, 376, 333-336 (1995)], IGF [Genes &Development, 15, 3023-8 (2003)] and the like.

The thus-obtained retinal tissue and retinal layer-specific neuron canbe identified by the presence or absence of the expression of a markergene and the like as an index, which may be combined as required. Theretinal layer-specific neuron obtained can also be identified byexamining the morphology of the cells. Furthermore, on the basis ofthese marker expression patterns or cell morphology, it is also possibleto isolate desired particular cells.

The expression of a marker gene can be confirmed by performingquantitative PCR as described in (2) above. Alternatively, the identitymay be confirmed by the expression of GFP and the like by manipulatingthe cells to allow a desired marker gene to be expressed as a fusionprotein of a marker gene product and GFP or the like. The expression ofthe protein may be detected using an antibody specific for a marker geneproduct.

Examples of useful marker genes include, but are not limited to,publicly known markers such as Rx (retinal progenitor cells), PAX6(retinal progenitor cells), nestin (expressed in hypothalamic neuronprogenitor cells, but not in retinal progenitor cells), Sox1 (expressedin hypothalamic nerve epithelium, but not in the retina), Crx(photoreceptor cell progenitor cells), Chx10 (bipolar cells and juvenileretinal progenitor cells), L7 (bipolar cells), Tuj1 (ganglion cells),Brn3 (ganglion cells), calretinin (amacrine cells), calbindin(horizontal cells), rhodopsin (photoreceptor cells), recoverin(photoreceptor cells), RPE65 (pigment epithelial cells), and Mitf(pigment epithelial cells). By combining as appropriate the presence andabsence of the expression of these marker genes, the cells obtained canbe identified. For example, amacrine cells are positive for bothcalretinin and Pax6 and negative for Tuj1, as stated above. Retinalganglion cells are positive for both Brn3 and Tuj1.

(4) Culture Products

The present invention also provides culture products as obtained by themethod of the present invention. The culture products of the presentinvention can include all of the cell culture products obtained by themethod of the present invention, such as a suspended aggregate of stemcells, cells obtained by dispersion-treating a suspended aggregate, andcells obtained by culturing dispersion-treated cells.

The culture products of the present invention also include homogenouscells and assembled cell populations isolated and purified from theabove-described culture products to the extent of acceptableadministration to subjects, for example, nerve progenitor cells (e.g.,retinal progenitor cells) and the like obtained via the steps (1) and(2), optic cup-like tissue, a mass of retinal progenitor cells obtainedvia the steps (1) and (2′), or retinal tissue, retinal layer-specificneuron and the like obtained via the steps (1), (2′) and (3).

The culture products of the present invention can be used as therapeuticdrugs for diseases based on disorders of nervous system cells (e.g.,retinal cells), as replenishers of cells and tissues injured by othercauses (e.g., for use in transplantation surgery), and for otherpurposes.

Examples of diseases based on disorders of nervous system cells includeParkinson's disease, spinocerebellar degeneration, Huntington chorea,Alzheimer's disease, ischemic cerebral diseases (e.g., cerebral stroke),epilepsy, brain traumas, spinal injuries, motor nerve diseases,neurodegenerative diseases, cochlear hearing loss, multiple sclerosis,amyotrophic lateral sclerosis, diseases caused by neurotoxic disorders,and the like. In particular, diseases based on retinal cell disordersinclude, for example, pigmentary degeneration of the retina, senilemacular degeneration, glaucoma, diabetic retinopathy, neonatalretinopathy, and retinal artery obstruction. The culture products of thepresent invention can also be used to supplement cells and tissues lostdue to ophthalmologic surgery (e.g., after retinoplasty for retinaldetachment) and the like (e.g., retinal transplantation surgery).

When using cells obtained by the method of the present invention (e.g.,nerve progenitor cells) as a therapeutic drug for a disease based on adisorder of the cells, it is preferable that the cells be transplantedto the subject after increasing the purity of the cells.

Any method of cell separation in public knowledge can be used for cellpurification. Such methods include, for example, a method using a flowcytometer [see, for example, Antibodies—A Laboratory Manual, Cold SpringHarbor Laboratory (1988), Monoclonal Antibodies: principles andpractice, Third Edition, Acad. Press (1993), Int. Immunol., 10, 275(1998)], the panning method [see, for example, Monoclonal Antibodies:principles and practice, Third Edition, Acad. Press (1993), AntibodyEngineering, A Practical Approach, IRL Press at Oxford University Press(1996), J. Immunol., 141, 2797 (1988)], and cell fractionation based onsucrose density differences [see, for example, Soshiki Baiyou noGijyutsu (3rd edition), Asakura Shoten (1996)].

The method of the present invention for increasing cell purity comprisesthe step of culturing cells obtained by differentiation-inducing theabove-described stem cells (e.g., nerve progenitor cells) in a mediumcontaining an anticancer agent. Thereby, undifferentiated cells can beremoved, making it possible to obtain differentiated cells of higherpurity, which are more suitable for pharmaceutical use. Hence, by atreatment with an anticancer agent, cells other than desireddifferentiated cells, for example, undifferentiated cells, can beremoved.

Here, the anticancer agent is exemplified by mitomycin C,5-fluorouracil, adriamycin, Ara-C, methotrexate and the like. Theseanticancer agents are preferably used at concentrations that are morecytotoxic to undifferentiated cells than to differentiation-inducedcells. Specifically, cultivation with these anticancer agents may beperformed in accordance with the above-described procedures ofcultivation to determine optimum concentrations. For example, a methodis useful wherein cells are cultured in a CO₂ incubator aerated with 5%gaseous carbon dioxide at 37° C. for several hours, preferably for 2hours, using a medium containing these anticancer agents atconcentrations one-hundredth to 1 time the range of concentrations forliving organisms specified in the Japanese Pharmacopoeia.

Any medium allowing cultivation of the differentiation-induced cells canbe used here. Specifically, the aforementioned media and the like areuseful.

In transplantation therapy, graft rejection due to histocompatibilityantigen differences is often problematic, which problem, however, can besolved by using a stem cell having the nucleus of a somatic celltransplanted thereto, or a stem cell having a modified gene on thechromosome thereof.

By inducing differentiation using a stem cell having the nucleus of asomatic cell transplanted thereto, nerve progenitor cells, retinalprogenitor cells, nervous system cells, retinal layer-specific neuronsand the like of the individual which is the donor of the somatic cellcan be obtained. Cells of such an individual are not only effective intransplantation medicine as they are, but also useful as a diagnosticmaterial for determining whether an existing drug is effective on theindividual. Furthermore, by culturing differentiation-induced cells fora long period, it is possible to determine their susceptibility tooxidative stress and senescence. By comparing their functions or lifespan with those of cells from other individuals, it is possible toevaluate the individual risks of contracting neurodegenerative and otherdiseases. These evaluation data are useful in providing an efficientprophylactic method for diseases diagnosed as developing at highincidences in the future.

Cells differentiation-induced from a stem cell by the method of thepresent invention, for example, nerve progenitor cells, retinalprogenitor cells, nervous system cells, retinal layer-specific neuronsand the like can be transplanted to a diseased site of a patient by amethod known per se [see, e.g., Nature Neuroscience, 2, 1137(1999)].

(5) Formation of Retinal Nerve Network

The present invention provides a method of allowing a retinal nervenetwork to be self-formed in vitro, comprising the steps (1), (2′) and(3). According to this method, it is possible to allow a cell aggregateobtained by the modified SFEBq method to form a retinal nerve networktherein without becoming a disarrayed nerve cell mass.

The construction of a retinal nerve network in the in vitro cellaggregates can be confirmed by, for example, observing electricalexcitement by light stimulation [Homma et al., (2009), J. Neurosci. Res.87(9)2175-2182] or imaging analysis with calcium release as an index.Here, “in vitro” refers to being not in a living organism.

In the retinal nerve network self-formed by the method of the presentinvention, an elevation of Ca²⁺ (calcium oscillation) synchronized ornon-synchronized with surrounding cells is repeatedly observed in manycells. Hence, the retinal nerve network formed by the method of thepresent invention preferably can be accompanied by a synchronizedspontaneous firing. Here, “firing” refers to an excitatory activity dueto depolarization of nerve cells, and “spontaneous firing” refers to thespontaneous occurrence of the firing. Hence, the retinal nerve networkformed by the method of the present invention can cause nerve activitiessimilar to the living retina in a certain aspect.

Provided according to the present invention is a culture product asobtained by the method of the present invention, specifically a cellaggregate that constitutes the above-described retinal nerve network.This culture product (cell aggregate) has formed a retinal nerve networkthat is extremely similar to the retinal nerve network in a livingorganism, so that it can be used for screening for therapeutic drugs fordiseases based on disorders of nervous system cells, for example,retinal cells, screening for therapeutic drugs for cell injuries due toother causes, or toxicity tests thereof and the like. Here, examples ofdiseases based on disorders of retinal cells include organic mercurypoisoning, chloroquine retinopathy, pigmentary degeneration of theretina, senile macular degeneration, glaucoma, diabetic retinopathy,neonatal retinopathy, and the like.

This culture product (cell aggregate) can also be used as a therapeuticdrug for diseases based on retinal cell disorders, a therapeutic drugfor cell injuries due to other causes and the like.

(6) Formation of Retinal Tissue Structure

The present invention provides a method of allowing the steric structureof retinal tissue to be self-formed in vitro, comprising the steps (1),(2′) and (3). According to this method, it is possible to form thesteric structure of retinal tissue in a cell aggregate obtained by themodified SFEBq method without becoming a disarrayed nerve cell mass.More preferably, it is possible to mimic the initial process of retinalhistogenesis with ongoing self-assembly in the same sequence as theretinal formation found in the optic cup primordium.

The self formation of the steric structure of retinal tissue in the cellaggregate in vitro can be confirmed by, for example, the expression oflayer-specific retinal cell markers such as Chx10, Tuj1, caltetinin,calbindin, and rhodopsin, light or electron microscopic morphologicalanalysis, live imaging of GFP-transferred cells and the like. Here, “invitro” has the same meaning as the above.

According to the present invention, the culture product obtained by themethod of the present invention, specifically an aggregate of cellsconstituting the steric structure of retinal tissue is provided. Theculture product of the present invention has a structure that ismorphologically extremely similar to the living retina, so that it canbe used for screening for therapeutic drugs for diseases based ondisorders of nervous system cells, particularly retinal progenitor cellsand retinal cells, screening for therapeutic drugs for cell injuries dueto other causes, or toxicity tests thereof and the like. Here, diseasesbased on disorders of retinal progenitor cells or retinal cells include,for example, organic mercury poisoning, chloroquine retinopathy,pigmentary degeneration of the retina, senile macular degeneration,glaucoma, diabetic retinopathy, neonatal retinopathy, and the like.

(7) Screening Method

The present invention provides a test substance screening methodcomprising using a culture product of the present invention.Particularly, a culture product of the present invention has an alreadyformed nerve network that is extremely similar to the living nervenetwork, and also has an already formed retinal tissue that is extremelysimilar to the histogenetic protrusion of the retina, so that it can beapplied for screening for therapeutic drugs for diseases based ondisorders of nervous system cells, for example, retinal progenitor cellsand retinal cells, screening for therapeutic drugs for cell injuries dueto other causes, or toxicity tests thereof, and development of a newtherapeutic method for diseases of nervous systems and the like.

Here, “a test substance” is exemplified by substances whose efficacy astherapeutic drugs for diseases of nervous systems is to be determinedand substances that are therapeutic drugs for other diseases whoseinfluences (e.g., toxicity) on nerves must be determined. The substancemay be any one of low-molecular compounds, high-molecular compounds,proteins, nucleic acids (DNA, RNA and the like), viruses and the like.Such substances can be chosen as appropriate by those skilled in theart.

The present invention is hereinafter described in more detail by meansof the following Comparative Examples and Examples, which, however, arefor illustrative purposes only and never limit the scope of theinvention.

EXAMPLES Comparative Example 1: Highly Efficient DifferentiationInduction into Cerebral Cortex Progenitor Cells by the SFEBq Method(Method)

EB5 cells of mouse ES cells (E14-derived) or cells of an E14-derivedcell line wherein the Venus gene, which is a modified GFP (greenfluorescent protein), had been knocked in the cerebral nerve marker Bf1gene as a nerve differentiation reporter by homologous recombination(hereinafter described as “Bf1/Venus-mES cells”) were cultured asdescribed in the literature (Watanabe et al., Nature Neuroscience,2005), and used in the experiments.

The medium for maintenance culture used was G-MEM medium (Invitrogen)supplemented with 1% fetal calf serum, 10% KSR (Knockout SerumReplacement; Invitrogen), 2 mM glutamine, 0.1 mM non-essential aminoacids, 1 mM pyruvic acid, 0.1 mM 2-mercaptoethanol and 2000 U/ml LIF.For nerve differentiation induction by suspension culture, ES cells weremono-dispersed using 0.25% trypsin-EDTA (Invitrogen), and suspended in150 μl of differentiation medium on a non-cell-adhesive 96-well cultureplate (SUMILON Spheroid plate, Sumitomo Bakelite Co., Ltd.) at 3×10³cells per well to allow aggregates to be formed quickly, after which theplate was incubated at 37° C., 5% CO₂ for 7 days (SFEBq method; FIG.1A).

The differentiation medium used in this operation was a serum-freemedium prepared by adding 10% KSR, 2 mM glutamine, 1 mM pyruvate, 0.1 mMnon-essential amino acids, 0.1 mM 2-ME, 250 μg/ml recombinant humanDkk-1, and 1 μg/ml recombinant human Lefty-1 to G-MEM medium (seeWatanabe et al., Nature Neuroscience, 2005).

The aggregate masses were recovered in a 6 cm non-adhesive plastic dish(3.5 ml of differentiation medium), and continued to besuspension-cultured for 3 days (10 days in total), after which thedifferentiation status was analyzed by fluorescent immunostaining. Theresults are shown in FIGS. 1A-1O.

(Results)

Immunostaining analysis revealed that 10 days after the start ofdifferentiation culture, about 70% of the cells in the aggregateexpressed the cerebrum-specific marker Bf1, with 90% of the Bf1-positivecells expressing the cerebral cortex-specific marker Emx1. Also whendifferentiated Bf1/Venus-mES cells were analyzed by the expression ofVenus-GFP, about 70% of the cells were positive, the majority of whichexpressed Emx1 (FIG. 1A). Hence, the SFEBq method enables cerebralcortex cells (progenitor cells) to be differentiation-induced with highefficiency when using the above-described differentiation medium. Whenusing a conventional method wherein aggregates of ES cells are graduallyformed using a 10 cm culture dish (Watanabe et al., Nature Neuroscience,2005), Bf1-positive cells accounted for up to 30%, of which less than40% became positive for cerebral cortex marker Emx1. The presence of anepithelium-like structure with polarity in the aggregates was confirmedby the expression of N-cadherin (Ncad), CD-133, laminin and the like(FIG. 1B to G; Dapi shows nucleus), electron microscopic observation ofthe morphology of tight junction (FIG. 1H, parenthesized), adherencejunction (FIG. 1I, parenthesized) and the like, rosette formation (FIG.1J, FIG. 1K, dotted line indicates a rosette), the expression ofpolarity markers and differentiation markers (FIGS. 1L to O, dotted lineindicates a rosette, asterisk indicates a lumen) and the like.

Hence, the SFEBq method, compared with the conventional method, promotesthe differentiation of ES cells into the cerebrum, particularly intocerebral cortex, more efficiently.

Comparative Example 2: In Vitro Production of Cerebral Neurons fromCerebral Cortex Progenitor Cells Induced by the SFEBq Method (Method)

Aggregates obtained by continued differentiation culture for 12 days bythe method described in Comparative Example 1 were enzymaticallydispersed (SUMILON Neural Tissue Dissociation kit), seeded onto aculture plate coated with poly-D-lysine/laminin/fibronectin at 5×10⁴dells/cm², and cultured using DMEM/F12 medium supplemented with 1×N2supplement and 10 ng/ml FGF2 for 2 days. Subsequently, the cells werefurther cultured using Neurobasal medium (supplemented with B27supplement)+50 ng/ml BDNF+50 ng/ml NT3 for 6 days. The properties of thedifferentiated neuron were analyzed by a fluorescent immunostainingmethod. The results are shown in FIGS. 2A-2F.

(Results)

Most of the cells in the test tube became TuJ1-positive neurons, ofwhich 80% were positive for the cerebral cortex-specific marker Emx1 andpositive for the glutamatergic neuron (abundantly present in cerebralcortex) marker VGluT1 (FIGS. 2A to B). Also observed was the expressionof a plurality of nerve markers characteristic of cerebral neuron(Telencephalin, GluR1, CamKII, Ctip2, Tbr1, Synapsin and the like)(FIGS. 2C to F).

Hence, differentiation of cerebral cortex progenitor cells induced bythe SFEBq method into cerebrum-specific neuron was confirmed.

Example 1: Highly Efficient Differentiation Induction to RetinalProgenitor Cells by the Modified SFEBq Method with High Concentrationsof Matrix Components Added and Optimized KSR Concentration (Method)

Rx-EGFP mES cells (mouse ES cells having EGFP knocked in at the earlyretinal progenitor cell marker gene Rx locus; Wataya et al, PNAS, 2008)were treated by the SFEBq method (a 96-well culture plate of lowcell-binding ability) to quickly generate homogenous aggregates at 3000cells per well, which were cultured for differentiation. Thedifferentiation induction medium used here was G-MEM medium(Invitrogen), 2% KSR, 2 mM glutamine, 0.1 mM non-essential amino acids,1 mM pyruvic acid and 0.1 mM 2-mercaptoethanol. Starting one day later,Matrigel was added to the medium in a ratio by volume of 1/100 to 1/25,and the cells were subjected to suspension aggregate culture for 7 days5% CO₂, at 37° C. Subsequently, the cultivation was further continuedunder the nerve differentiation promoting conditions. Differentiationanalysis was performed by the fluorescent immunostaining method (frozensections) using the brain nerve progenitor marker Sox1, the cerebralmarker Bf1, the retinal progenitor cell marker Rx, the neural retinalprogenitor cell and bipolar cell marker Chx10, the photoreceptor cellmarker rhodopsin antibody and the like.

(Results)

When using the medium comprising a 1/100 volume of Matrigel supplementedwith 10% KSR as in Comparative Example 1, cerebral cortex progenitorcells expressing Bf1 were induced with constant efficiency (>50%; 9 dayslater). Meanwhile, when using the same medium but supplemented with 2%KSR under the same conditions, the expression of Bf1 decreased to lessthan 10%. With the 2% KSR medium comprising a 1/50 or 1/25 volume ofMatrigel, the expression of Bf1 became less than 5%.

Conversely, in differentiation culture with a 2% KSR medium supplementedwith Matrigel in a 1/100 volume or more, Rx-EGFP-positive and Rxantibody-positive cells emerged at 5% or more of the cells in the cellmasses. With the addition of Matrigel in a 1/50 or 1/25 volume or more,about 60% of the cells became Rx-EGFP-positive. In differentiationculture with a 10% KSR medium supplemented with Matrigel in a 1/100volume, the expression of Bf1 was less than 1%.

Example 2: Self Formation of Optic Cup-Like Tissue from RetinalProgenitor Cells Using the Modified SFEBq Method (Method)

The aggregates of Rx-positive cells obtained in Example 1 were culturedusing a culture broth prepared by adding Matrigel in a 1/50 volume to 2%KSR medium for 7 days, after which it was transferred to a culture brothprepared by adding an N2 additive to the DMEM/F12 medium, and furthersuspension-cultured under 5% CO₂/40% O₂ conditions for 3 days.

(Results)

The strongly Rx-EGFP-positive portion formed a tissue as a protrusionfrom the cell mass (FIG. 3). Its histological profile was similar tothat of the fetal optic cup (an early retinal tissue formed as aprotrusion from the diencephalon), representing a structure whereinRx-positive, Chx10-positive juvenile neural retinal tissue was wrappedby a layer of retinal pigment cells.

Example 3: Self Assembly of Retinal Tissue from Retinal Progenitor CellsUsing the Modified SFEBq Method (Method)

Optic cup-like tissue as obtained in Example 2 (10 days of culture) wasseparated from the cell mass using microtweezers, and subjected tosuspension culture with DMEM/F12/N2+0.5 μM retinoic acid (known topromote the survival of photoreceptor cells).

(Results)

The suspension-cultured optic cup-like tissue had been induced to alaminar structure that is structurally extremely similar to the retinaafter birth (FIGS. 2A-2F and 3). Regularly formed in the outermost layerwas a planar structure of photoreceptor cells (rhodopsin-positive andhaving an outer segment structure), possessing the right cell polarity.Under this layer were a layer of Chx10-positive bipolar cells and alayer of Calbindin-positive horizontal cells, under which a layer ofCalretinin-positive/Pax6-positive/TuJ1-negative amacrine cells waspresent, and the lowermost layer was a layer ofBrn3-positive/TuJ1-positive retinal ganglion cells, which were formed inan orderly manner (FIG. 5). The order of these layers corresponded tothe retinal laminar structure in vivo. In summary, it has been shownthat when applying the modified SFEBq method for suspension cell massculture by the SFEBq method in consideration of the optimizedcombination of matrix treatment and medium, not only retinal progenitorcells are formed efficiently, but also a retinal tissue having a laminarstructure is self-formed in vitro from these retinal progenitor cells.

Example 4: Electrophysiological Activities of Retinal Tissue Produced bythe Modified SFEBq Method (Method)

Optic cup-like tissue was separated from the cell mass usingmicrotweezers in the same manner as Example 3, and cultured withDMEM/F12/N2+0.5 μM retinoic acid. For electrophysiological examination,the tissue was cultured on a plate with multiple planar microelectrodes(MED probe); 2 days later, the action potentials of the axons emergingfrom the optic cup-like tissue were examined by the multipolar electrodefield potential method (MED64; Alpha MED Scientific Inc.).

(Results)

The axons from the optic cup-like tissue were Tuj1-positive and thoughtto be derived from ganglion cells same as retinal tissue. A large numberof spontaneous firings of irregular action potentials from these axonswere observed by the multipolar electrode field potential method. Theseresults confirmed that a network that induces spontaneous nerveactivities, observed in juvenile retina of newborn babies in vivo andthe like, had been formed in the optic cup-like tissue. It seems alsopossible to examine light-induced action potentials with MED 64 by themethod of Homma et al. [Homma et al, (2009), J. Neurosci. Res.87(9)2175-2182].

Example 5: Spontaneous Formation of Optic Cup-Like Protrusion Tissuefrom Human ES Cells (Method)

Human ES cells (khES1) were cultured for maintenance by an ordinarymethod (Ueno et al, PNAS 103, 9554-9559, 2006). The human ES cells wereisolated from the plate by a method already in the public domain, andmono-dispersed with trypsin (Watanabe et al., Nature Biotech. 25,681-686, 2007). These cells were quickly re-aggregated using a 96-wellculture plate of low cell-binding ability in the same way as ComparativeExample 1 and Example 1 to obtain homogenous aggregates. In thatoperation, the cells were suspended in a culture broth at 9000 cells perwell of the 96-well plate. The culture broth used was DMEM/F12+10-20%KSR+2 mM glutamine+0.1 mM non-essential amino acids+0.1 mM 2-ME, withthe addition of 10 μM of the Rock inhibitor Y-27632 (a cell deathsuppressant) during the first 6 days. Starting at 3 days of cultivation,Matrigel was added in a 1/100 volume, and the cells were cultured untilday 18. Between day 18 and day 25, suspension culture was continuedusing DMEM/F12+N2+1 μM RA, with the O₂ concentration raised to 40%.Between day 25 and day 40, the cells were suspension-cultured in thepresence of Neurobasal+B27+1 μM RA, 40% O₂.

(Results)

In the above-described cultivation, only when Matrigel was added, theformation of a continuous epithelial tissue of Rx-positive,Pax6-positive retinal progenitor cells was observed in a cell massderived from a human ES cell. After 20 days of cultivation, as on day 7of mouse ES cell culture, the formation of Rx-positive, Pax6-positivetissue as a protrusion from the main body of a cell mass derived fromthe human ES cell was confirmed. They comprised strongly Rx-positiveneural retina progenitor tissue and weakly Rx-positive pigmentepithelium progenitor tissue, both of which were nestin-negative. Also,35 days later, the presence of Rx-positive, Pax6-positive,nestin-negative pseudostratified columnar epithelium tissue (amorphological characteristic of neural retina progenitor tissue) wasconfirmed.

Example 6: Efficient Induction of Retinal Epithelium in ES CellAggregates Using Purified Matrix Proteins and Nodal/Activin (Method)

Rx-GFP ES cells (3000 cells/well, 96-well plate) were cultured in SFEBqculture with G-MEM supplemented with 1.5% KSR. In this experiment,instead of adding Matrigel to culture, purified laminin and entactin(High concentration Laminin/Entactin complex; BD; 120 μg/ml) were addedas extracellular matrix proteins on day 1 (24 hours after the onset ofdifferentiation culture). Recombinant Mouse Nodal (R&D; 500-1000 ng/mL)or Human Activin (R&D; 250 ng/ml) was also added on day 1 and the Nodaltreatment was continued until day 7. SFEBq aggregates were cultured for7-10 days and the formation of RxGFP+ vesicles and cup structures wereexamined under a fluorescent microscope. Nodal and Activin are known toact on common cell surface receptors and activate Smad2/3 signals in thecell.

Results:

ES cells cultured without extracellular matrix proteins (Matrigel orlaminin/entactin) or Nodal did not express Rx-GFP on days 7-10. Unlike2% Matrigel, laminin+entactin alone did not induce Rx-GFP+ retinalepithelium on day 7 or 10. In contrast, when cells were treated withlaminin+entactin and Nodal (both 500 and 1000 ng/ml) or Activin duringdays 1-7, large patches of Rx-GFP+ epithelia appeared in the SFEBqaggregates on day 7 (FIG. 7). They formed optic vesicle-like sacs on day7 and later exhibited the optic cup-like morphology on day 10. TGF-beta1or 2 (1000 ng/ml) did not replace the inducing activity of Nodal evenwhen combined with laminin+entactin. Treatment of SFEBq cells with Nodalor Activin from day 0 inhibited both neural (Sox1) and retinal (Rx)differentiation in accordance with previous reports (Watanabe et al,Nature Neuroscience, 2005), indicating that the absence of Nodal/Activinsignals at the initial phase of SFEBq culture is a preferred condition.

The three-dimensional retinal tissue formation can be induced by thedefined matrix proteins laminin and entactin in the presence of Nodal.

While the present invention has been described with emphasis onpreferred embodiments, it is obvious to those skilled in the art thatthe preferred embodiments can be modified. The present invention intendsthat the present invention can be embodied by methods other than thosedescribed in detail in the present specification. Accordingly, thepresent invention encompasses all modifications encompassed in the gistand scope of the appended “CLAIMS.”

The contents disclosed in any publication cited herein, includingpatents and patent applications, are hereby incorporated in theirentireties by reference, to the extent that they have been disclosedherein.

INDUSTRIAL APPLICABILITY

According to the method of the present invention, it is possible toefficiently differentiation-induce nervous system cells, thus allowingcytotherapy to be applied for neurodegenerative diseases. The method ofthe present invention also makes it possible to efficientlydifferentiation induction of diencephalon tissues (particularly retinaltissue), a task that has been difficult to achieve by the conventionalmethod of differentiation, thus allowing cytotherapy to be applied fordiseases associated with abnormalities of diencephalon tissues(particularly retinal tissue).

Furthermore, according to the present invention, the steric structure ofretinal tissue having a retinal nerve network and a laminar structurecan be produced in vitro. Therefore, the present invention is also ofhigh utility in providing “tissue materials” that serve well inregenerative medicine, drug discovery and toxicity tests for theabove-described pharmaceuticals and the like.

Another advantage of the present invention is that the risk in thetransplantation of cells obtained by stem cell culture can be reduced tothe risk levels in allotransplantation because it does not involve theuse of an animal-derived cell as an inductor.

This application is based on a patent application No. 61/258,439 filedin United States, the contents of which are incorporated in full hereinby this reference.

1. A cell aggregate comprising Rx-positive cells and having been inducedin vitro to differentiate from a pluripotent stem cell, wherein aRx-positive portion forms a protrusion from the non-retinal rest of theaggregate, and wherein the non-retinal rest of the aggregate is notdiencephalon.
 2. The aggregate according to claim 1, wherein theprotrusion comprises Rx-positive, Chx10-positive juvenile neural retinaltissue.
 3. The aggregate according to claim 1, wherein the aggregatecomprises a plurality of the protrusions.
 4. The aggregate according toclaim 1, wherein the aggregate comprises a continuous epithelial tissueof Rx-positive, Pax6-positive retinal progenitor cells.
 5. The aggregateaccording to claim 1, wherein the protrusion comprises Rx-positive,Pax6-positive tissue.
 6. The aggregate according to claim 1, wherein theprotrusion comprises strongly Rx-positive neural retina progenitortissue and weakly Rx-positive pigment epithelium progenitor tissue, bothof which are nestin-negative.
 7. The aggregate according to claim 1,wherein the aggregate comprises Rx-positive cells and non-Rx-positivecells, and wherein a Rx-positive portion forms the protrusion from themain body of the cell aggregate and the non-retinal rest of theaggregate is non-Rx-positive and comprises Bf1 positive cells.
 8. Aretinal tissue with a laminar structure which has a layer ofChx10-positive bipolar cells and a layer of Calbindin-positivehorizontal cells under the outermost layer.
 9. The retinal tissueaccording to claim 8, wherein a planer structure of photoreceptor cells,which is rhodopsin-positive and having an outer segment structure, isformed in the outermost layer, possessing the right cell polarity. 10.The retinal tissue according to claim 8, which has a layer ofCalretinin-positive/Pax6-positive/TuJ1-negative amacrine cells under thelayer of Calbindin-positive horizontal cells, and the lowermost layer isa layer of Brn3-positive/TuJ1-positive retinal ganglion cells, which areformed in an orderly manner.
 11. A method of producing a retinal tissuewith a laminar structure which has a layer of Chx10-positive bipolarcells and a layer of Calbindin-positive horizontal cells under theoutermost layer, comprising the steps: (1) separating the protrusionfrom the aggregate described in claim 1, and (2) subjecting theseparated protrusion to suspension culture.